Network-based integration of mRNA and miRNA profiles reveals new target genes involved in pancreatic cancer.

Pancreatic cancer is regarded as the most fatal and aggressive malignancy cancer due to its low 5-year survival rate and poor prognosis. The approaches of early diagnosis and treatment are limited, which makes it urgent to identify the complex mechanism of pancreatic oncogenesis. In this study, we used RNA-seq to investigate the transcriptomic (mRNA and miRNA) profiles of pancreatic cancer in paired tumor and normal pancreatic samples from ten patients. More than 1000 differentially expressed genes were identified, nearly half of which were also found to be differentially expressed in the majority of examined patients. Functional enrichment analysis revealed that these genes were significantly enriched in multicellular organismal and metabolic process, secretion, mineral transport, and intercellular communication. In addition, only 24 differentially expressed miRNAs were found, all of which have been reported to be associated with pancreatic cancer. Furthermore, an integrated miRNA-mRNA interaction network was generated using multiple resources. Based on the calculation of disease correlation scores developed here, several genes present in the largest connected subnetwork, such as albumin, ATPase H+ /K+ exchanging alpha polypeptide and carcinoembryonic antigen-related cell adhesion molecule 1, were considered as novel genes that play important roles in the development of pancreatic cancer. Overall, our data provide new insights into further understanding of key molecular mechanisms underlying pancreatic tumorigenesis.

Risk factors and managements of hemorrhage associated with pancreatic fistula after pancreaticoduodenectomy.

BACKGROUND: Post-pancreaticoduodenectomy pancreatic fistula associated hemorrhage (PPFH) is one of the leading lethal complications. Our study was to analyze the risk factors and managements of hemorrhage associated with pancreatic fistula after pancreaticoduodenectomy, and to evaluate treatment options. METHOD: We analyzed 445 patients who underwent pancreaticoduodenectomy or pylorus-preserving pancreaticoduodenectomy and evaluated the relevance between clinical data and PPFH. RESULTS: The incidence of postoperative pancreatic fistula (POPF) was 27.42% (122/445), and the incidence of PPFH was 4.49% (20/445). Among the 20 patients with PPFH, 7 died and 13 were cured. Interventional angiographic therapy was performed for 10 patients and 5 were successfully treated. Relaparotomy was performed for 5 patients and 2 were successfully cured. Univariate logistic regression analysis indicated that several risk factors were related to PPFH: the nature of tumor (carcinoid/low-grade or high-grade malignancy), preoperative day 1 serum prealbumin, preoperative day 1 total bilirubin (TBIL), operative time, blood loss in the operation, operative method (vascular resection and revascularization), postoperative day 3 TBIL, biliary fistula, and the grade of POPF. The multivariate stepwise logistic regression analysis demonstrated that the nature of tumor and the grade of POPF were independently risk factors of PPFH. Receiver operating characteristic curve indicated that preoperative day 1 serum prealbumin level <173 mg/L and postoperative day 3 TBIL level ≥168 µmol/L were the risk factors of PPFH. CONCLUSIONS: The risk of PPFH was found to be increased with high potential malignancy and high grade of POPF. Angiography-embolization is one of the major and effective therapies for PPFH. Extraluminal-intraluminal PPFH is more serious and needs more aggressive treatments.

Downregulation of ASPP2 in pancreatic cancer cells contributes to increased resistance to gemcitabine through autophagy activation.

BACKGROUND: Apoptosis-stimulating of p53 protein 2 (ASPP2) is one of the ASPP family members and it has been reported to be associated with human cancer. However, the role of it in pancreatic cancer is still not clear. METHODS: We analyzed the expression level of ASPP2 in cancer tissue samples with RT-qPCR, Western Blotting assay and immunohistochemistry staining. We studied the biological function of ASPP2 and its mechanism with gene overexpression and gene silencing technologies. We determined the sensitivity of pancreatic cells with differential ASPP2 level to gemcitabine and whether autophagy inhibition affected the gemcitabine resistance, both in vitro and in vivo. RESULTS: Expression of ASPP2 was downregulated in cancerous tissues in comparison with para-cancerous tissues. ASPP2 expression was linked to clinical outcomes in patients and down-regulation of ASPP2 increased cell proliferation, autophagic flux, the activity of AMP Kinase of pancreatic cancer cells and vice versa. Knockdown of ASPP2 results in increased resistance to gemcitabine, which was attributed to the enhanced autophagy. CONCLUSIONS: ASSP2 expression is lower in cancerous tissues and decreased ASPP2 lead to higher cancer cells proliferation and autophagic flux, which contribute to the gemcitabine resistance.

Sox2 function as a negative regulator to control HAMP expression.

BACKGROUND: Hepcidin, encoding by HAMP gene, is the pivotal regulator of iron metabolism, controlling the systemic absorption and transportation of irons from intracellular stores. Abnormal levels of HAMP expression alter plasma iron parameters and lead to iron metabolism disorders. Therefore, it is an important goal to understand the mechanisms controlling HAMP gene expression. RESULTS: Overexpression of Sox2 decrease basal expression of HAMP or induced by IL-6 or BMP-2, whereas, knockdown of Sox2 can increase HAMP expression, furthermore, two potential Sox2-binding sites were identified within the human HAMP promoter. Indeed, luciferase experiments demonstrated that deletion of any Sox2-binding site impaired the negative regulation of Sox2 on HAMP promoter transcriptional activity in basal conditions. ChIP experiments showed that Sox2 could directly bind to these sites. Finally, we verified the role of Sox2 to negatively regulate HAMP expression in human primary hepatocytes. CONCLUSION: We found that Sox2 as a novel factor to bind with HAMP promoter to negatively regulate HAMP expression, which may be further implicated as a therapeutic option for the amelioration of HAMP-overexpression-related diseases, including iron deficiency anemia.

Identifying Liver Metastasis-Related Genes Through a Coexpression Network to Construct a 5-Gene Model for Predicting Pancreatic Ductal Adenocarcinoma Patient Prognosis.

OBJECTIVES: This study aimed to develop a liver metastasis-related gene prognostic index (LMPI) for pancreatic ductal adenocarcinoma prognosis and therapy. METHODS: The Cancer Genome Atlas data set was used to identify liver metastasis-related hub genes via weighted gene coexpression network analysis. The core genes were identified to construct an LMPI by using the Cox regression method. An immune cell abundance identifier was applied to determine the immune cell abundance. RESULTS: A total of 78 hub liver metastasis-related genes in the black module were significantly enriched in complement and coagulation cascades, fat digestion and absorption, and the PPAR signaling pathway. Then, an LMPI was constructed on the basis of the 5 prognostic genes (MOGAT3, ASGR1, TRPM8, SGSM1, and LOC101927851). Patients with higher LMPI scores had poor overall survival, more co-occurring or mutually exclusive pairs of driver gene mutations, and less benefit from immunotherapy than patients with lower LMPI scores. In addition, a high correlation was also found between LMPI scores and immune infiltration, such as CD4 naive, CD8 T, cytotoxic T, T helper 2, follicular helper T, and natural killer cells. CONCLUSIONS: The core genes of the LMPI developed may be independent factors for predicting prognosis, immune characteristics, and immunotherapy efficacy in pancreatic ductal adenocarcinoma.

Silencing of hHS6ST2 inhibits progression of pancreatic cancer through inhibition of Notch signalling.

Many of the ligands involved in developmental processes require HS (heparan sulfate) to modulate signal transduction. hHS6ST2 (human heparan sulfate D-glucosaminyl 6-O-sulfotransferase-2) is a Golgi-resident enzyme that usually acts on GlcA/IdoA(2S)-GlcNAc/NS disaccharide-6-sulfate modifications within the HS sequence. Emerging evidence indicates the importance of 6-O-sulfation in a number of developmental processes. However, any correlation with cancer-related events remains largely unexplored. In the present study, we found that hHS6ST2, but not other variants, was activated in human PC (pancreatic cancer). shRNA (short hairpin RNA)-mediated silencing of endogenous hHS6ST2 expression in the PC cell line PANC-1 inhibited cell invasion and migration. hHS6ST2 knockdown also resulted in markedly reduced tumorigenesis in immunocompromised mice. To specifically explore the molecular alterations resulting from depletion of hHS6ST2-generated 6-O-sulfation, we employed two-dimensional gel electrophoresis technology followed by nano-HPLC-ESI (electrospray ionization)-tandem MS to separate and identify total proteins from PC cells. Our data suggest that hHS6ST2 potentiates Notch signalling in PC cells. We also identified a role for hHS6ST2 in the growth and tumorigenicity of these cells which, at least in part, acts through Notch-mediated EMT (epithelial-mesenchymal transition) and angiogenesis. The results of the present study suggest that hHS6ST2 could be an attractive target for PC therapy.

Association between preoperative prealbumin level and postoperative mortality and morbidity after hepatic resection for hepatocellular carcinoma: A multicenter study from a HBV-endemic area.

BACKGROUND: Prealbumin is a more sensitive serum biomarker in reflecting liver function and nutritional status than albumin, because of its shorter half-life and its characteristics that could hardly be affected by supplemental venous infusion of albumin or blood transfusion. This study aimed to identify whether preoperative prealbumin level was associated with postoperative mortality and morbidity after hepatic resection for patients with hepatocellular carcinoma (HCC). METHODS: From a Chinese multicenter database, patients who underwent hepatic resection for HCC were divided into the low and normal prealbumin groups by using 17 mg/dL as the cut-off level for serum prealbumin taken within a week before surgery. Using univariable and multivariable logistic regression analyses, independent predictors associated with postoperative 30-day and 90-day mortality, 30-day overall and major morbidity, and postoperative hepatic insufficiency were identified. RESULTS: Among 1356 patients, 409 (30.2%) had a low preoperative prealbumin level. Postoperative 30-day and 90-day mortality, and 30-day overall and major morbidity in the low prealbumin group were significantly higher than the normal prealbumin group (2.9% vs. 0.5%, 5.1% vs. 1.5%, 35.7% vs. 18.4%, and 14.4% vs. 6.5%, respectively, all P < 0.001). Multivariable analyses identified that preoperative prealbumin level, but not albumin level, was independently associated with postoperative 30-day mortality (OR: 3.486, 95% CI: 1.184-10.265), 90-day mortality (2.504, 1.219-5.145), 30-day overall morbidity (1.727, 1.302-2.292), 30-day major morbidity (1.770, 1.155-2.711) and postoperative hepatic insufficiency (1.967, 1.119-3.427). CONCLUSIONS: Preoperative prealbumin level could be used to predict postoperative morbidity and mortality for patients treated with hepatic resection for HCC.

[The role of full length superior mesenteric artery isolation in extended pancreaticoduodenectomy in the treatment of ductal adenocarcinoma of the pancreas in the uncinate process].

OBJECTIVE: To improve the prognosis and safety of extended pancreaticoduodenectomy for patients with pancreatic cancer in the uncinate process of pancreas. METHODS: From January 2004 to March 2008, 26 extended pancreaticoduodenectomies with full length superior mesenteric artery (SMA) isolation and mesentery root resection were performed for the ductal adenocarcinomas in the uncinate process of pancreas. There were 16 males and 10 females aging from 30 to 75 years old [medium age (55.0 +/- 13.0) years old]. Eleven of 26 patients were combined with portal vein-superior mesenteric vein resection. The effect and safety of this procedure were analyzed retrospectively. RESULTS: There was no operative mortality in all patients. The pathological examination showed that all the incisal margins were negative. After a follow-up of 7 to 45 months, the pain relief was occurred in all patients. The 1-year, 2-year accumulated survival rates were 72.2%, and 48.1%, respectively. CONCLUSIONS: Full length SMA isolation and the mesentery resection in extended pancreaticoduodenectomy are safe and effective. The procedure is also benefit for the patients in improving the survival rate and quality of life.

A novel scoring system predicts postsurgical survival and adjuvant chemotherapeutic benefits in patients with pancreatic adenocarcinoma: Implications for AJCC-TNM staging.

BACKGROUND: We evaluated the application of the latest 8th American Joint Committee on Cancer (AJCC) staging system in Chinese patients and determined whether the addition of biologic markers could improve the prediction of postsurgical survival in pancreatic adenocarcinoma (PC). METHODS: This multicenter study involved 1,223 consecutive patients who underwent margin-negative pancreatectomy for PC. A scoring system was devised based on AJCC pathologic parameters and biologic markers and defined using a Cox proportional hazards model. Prognostic accuracies were evaluated by concordance index (C-index) and Akaike information criterion (AIC). RESULTS: The 8th edition AJCC staging system had a better survival distribution of PC with different stages and a similar C-index (0.62 in the training cohort, 0.60 in the validation cohort) than the 7th edition (0.59 in the training cohort, 0.58 in the validation cohort). Nevertheless, survival of resected patients with stage IIA or IIB disease was indistinguishable. Incorporation of postoperative carbohydrate antigen 19-9 (CA19-9) levels and tumor grade into the 8th edition AJCC staging system generated a scoring system with better predictive accuracy for overall survival (OS) (C-index of 0.73 and AIC of 4301.05 in the training cohort, C-index of 0.71 and AIC of 3309.23 in the validation cohort). More importantly, patients with median or higher scores experienced OS benefits from adjuvant chemotherapy. CONCLUSION: Postoperative CA19-9 levels and tumor grade are two well-known PC biologic markers that could be incorporated into a standard AJCC staging system to refine risk stratification and predict OS benefit from adjuvant chemotherapy in resected PC.

HIF1/2α mediates hypoxia-induced LDHA expression in human pancreatic cancer cells.

Glycolysis is a typical conduit for energy metabolism in pancreatic cancer (PC) due to the hypoxic microenviroment. Lactate dehydrogenase A (LDHA) catalyzes the conversion of pyruvate to lactate and is considered to be a key checkpoint of anaerobic glycolysis. The aim of the present study was to explore the mechanism of interactions between hypoxia, HIF-1/2α and LDHA, and the function of LDHA on PC cells by analyzing 244 PC and paratumor specimens. It was found that LDHA was over-expressed and related to tumor stages. The result of in vitro study demonstrated that hypoxia induced LDHA expression. To explore the relationship between HIF and LDHA, chromatin immunoprecipitation assay and luciferase assay were performed. The result showed that HIF-1/2α bound to LDHA at 89bp under the hypoxic condition. Furthermore, knockdown of endogenous HIF-1α and HIF-2α decreased the LDHA expression even in the hypoxic condition, which was accompanied with a significant decrease in lactate production and glucose utilization (p < 0.01). Immunofluorescence in the 244 specimens showed that HIF-1/2α was over-expressed and associated with LDHA over-expression (p < 0.0001). Forced expression of LDHA promoted the growth and migration of PC cells, while knocking down the expression of LDHA inhibited the cell growth and migration markedly. In summary, the present study proved that HIF1/2α could activate LDHA expression in human PC cells, and high expression of LDHA promoted the growth and migration of PC cells.

[Clinicopathological study of lymphatic vessel density and distribution in pancreatic cancer].

OBJECTIVE: To clarify the clinicopathological significance of lymphatic vessel density (LVD) and distribution in pancreatic cancer. METHODS: We measured LVD in 43 pancreatic cancer specimens by immunostaining with specific lymphatic endothelium marker, and examined their relationship with well-defined clinicopathological variables. RESULTS: Intratumoral LVD (9.4 +/- 10.0) was significantly lower than periturmoral (16.0 +/- 9.7) (P < 0.001) and nontumoral LVD (13.5 +/- 6.0) (P < 0.01). Increased peritumoral LVD correlated significantly with tumor staging (P < 0.05) and lymph node involvement (P < 0.05). CONCLUSION: The lymphatic vessels distribution in pancreatic cancer samples and peritumoral lymphangiogenesis may promote the malignant progression and lymph node metastasis of pancreatic cancer.

Ulinastatin reduces the resistance of liver cancer cells to epirubicin by inhibiting autophagy.

During chemotherapy, drug resistance caused by autophagy remains a major challenge to successful treatment of cancer patients. The purpose of this study is to show that ulinastatin (UTI), a trypsin inhibitor, could reduce the resistance of liver cancer cells to chemotherapeutic agent epirubicin (EPI). We achieved this conclusion by analyzing the effect of EPI alone or UTI plus EPI on SMMC-7721 and MHCC-LM3 liver cancer cells. We also generated an EPI-resistant liver cancer cell line (MHCC-LM3er cells), and found that UTI could sensitize the LM3er cells to EPI. Autophagy usually functions to protect cancer cells during chemotherapy. Our study showed that UTI inhibited the autophagy induced by EPI in liver cancer cells, which promoted apoptosis, and therefore, reduced the resistance of the cancer cells to EPI. Further studies showed that the UTI-mediated inhibition on autophagy was achieved by inhibiting transcriptional factor nuclear factor-κB (NF-κB) signaling pathway. To verify our results in vivo, we injected MHCC-LM3 liver cancer cells or EPI-resistant LM3er cells into mice, and found that EPI could only effectively inhibit the growth of tumor in MHCC-LM3 cell-injected mice, but not in LM3er cell-injected mice. However, when UTI was also administered, the growth of tumor was inhibited in the MHCC-LM3er cell-injected mice as well. Our results suggest that UTI may be used in combination with anti-cancer drugs, such as EPI, to improve the outcome of cancer therapy.

[Clinical significance of regional lymphadenectomy in radical resection of ductal adenocarcinoma in the pancreatic head].

OBJECTIVE: To prospectively evaluate the long-term effect of pancreaticoduodenectomy with regional lymphadenectomy. METHODS: One hundred and twenty-one patients with ductal adenocarcinoma in the pancreatic head treated from 1996 to 2001 were studied prospectively. The enrollment of the patients was dependent on 7 criteria. The patients were divided into two groups: regional lymphadenectomy (group A, n = 50) and routine Whipple procedure (group B, n = 71). Their pre- and postoperative conditions, clinicopathological data, survival rates were studied. RESULTS: It was comparable between the 2 groups in age, sex, preoperative risk factors, operative management, and postoperative complication. Clinicopathological results showed no difference in tumor size and plexus invasion; but the frequency of lymph node involvement and the amount of resected lymph node in group A were significantly higher than those in group B. The rate of local recurrence was significantly higher in group A than in group B. The survival rates of 1-, 3-, 5-year in group A were 70.8%, 31.4%, 20.9%, respectively, which were higher than those in group B. No direct relations were observed between nodal involvement and survival rate. CONCLUSION: Lymphadenectomy in radical pancreaticoduodenectomy could remove lymph nodes effectively and sufficiently, and reduce the rate of local recurrence so as to improve the long-term survival rate.

Sox2 function as a negative regulator to control HAMP expression

BACKGROUND: Hepcidin, encoding by HAMP gene, is the pivotal regulator of iron metabolism, controlling the systemic absorption and transportation of irons from intracellular stores. Abnormal levels of HAMP expression alter plasma iron parameters and lead to iron metabolism disorders. Therefore,itis animportant goal to understand the mechanisms controlling HAMP gene expression. RESULTS: Overexpression of Sox2 decrease basal expression of HAMP or induced by IL-6 or BMP-2, whereas, knockdown of Sox2 can increase HAMP expression, furthermore, two potential Sox2-binding sites were identified within the human HAMP promoter. Indeed, luciferase experiments demonstrated that deletion of any Sox2-binding site impaired the negative regulation of Sox2 on HAMP promoter transcriptional activity in basal conditions. ChIP experiments showed that Sox2 could directly bind to these sites. Finally, we verified the role of Sox2 to negatively regulate HAMP expression in human primary hepatocytes. CONCLUSION: We found that Sox2 as a novel factor to bind with HAMP promoter to negatively regulate HAMP expression, which may be further implicated as a therapeutic option for the amelioration of HAMP-overexpression-related diseases, including iron deficiency anemia.